KR20200117659A - New yeast Debaryomyces hansenii KD2 strain isolated from Korean traditional fermented soybean foods and its use - Google Patents

Abstract

The present invention relates to a novel fermented yeast strain Debaryomyces hansenii KD2 which is isolated from Korean traditional fermented soybean paste, and a use thereof. The novel fermented yeast strain Debaryomyces hansenii KD2 which is isolated from Korean traditional fermented soybean paste has a structure in which a charcoal coating liquid (20) obtained by mixing ceramics, charcoals, and PVA with a Dendropanax morbifera extract is coated on the surface of a metallic kitchenware main body (10), and a loess coating liquid (30) obtained by mixing ceramics, loess, and PVA with a loess extract is coated on the surface of the charcoal coating liquid (20). A triglyceride level is lowered, and good cholesterol is improved.

Description

New yeast Debaryomyces hansenii KD2 strain isolated from Korean traditional fermented soybean foods and its use}

The present invention relates to the development of novel traditional soybean fermented yeast strains and probiotic yeast strains using the same, and more specifically, discovery and identification of Debaryomyces sp. in traditional Korean soybeans , growth, genome, and The characteristics of flavor profile and ability to modulate immune activity, and methods of utilizing traditional soybean fermentation using the same.

In order to manufacture traditional Korean soybean paste, soaked beans are boiled and then molded into square meju and hung in the air for several months using rice straw for natural fermentation. Then, soak the meju in salt water and add soybean paste and soy sauce. During the fermentation process of meju, various microorganisms such as bacteria, yeast, and fungi are involved, and proteins are decomposed and flavor components are produced by various enzymes that they produce. Therefore, Korean traditional paste has various flavors and flavors depending on the region where it is produced due to the complex fermentation activity of various microbial strains that exist naturally. In the case of Korean traditional paste based on fermentation by microorganisms, various bacteria, yeast, and fungal strains are involved, and fungi and yeast strains play a key role in the process of decomposing proteins, starches, and fats into amino acids, glucose, and fatty acids. And wild yeast and lactic acid bacteria create the taste and aroma of paste. The identification of strains of microbial populations living in traditional soybeans is a comparison of 16S rRNA for bacteria, 5.8S rRNA for yeast/fungi, or ITS (Internal transcribed spacer) gene sequence comparison and PCR-denaturing gradient gel electrophoresis. ; DGGE) analysis-based classification method is being used, and recently, an analysis study on the entire microbial community living in Meju and Doenjang using Next Generation Sequencing (NGS) technique has been attempted. Studies on Bacillus and lactic acid bacteria, which are known to exist as dominant species in Korean traditional soybean paste, are being actively conducted, and through studies on the effect of enzyme activity derived from fungi and fungi on the aroma components of soybean paste, the type of meju fungus, enzyme activity in meju, and maturation Studies are being conducted to analyze the correlation between the period and the fragrance components of doenjang, and to identify the main factors for controlling and improving the flavor of doenjang. Among some traditional Korean soybeans, Pichia burtonii , Pichia farinosa , Candida rugosa , Yeast strains such as Zygosaccharomyces rouxii have also been identified as dominant species, but studies on fermentation physiology and function of yeast strains derived from Meju are poor compared to fungal strains. Meanwhile, in the case of Japanese soy sauce and miso, various types of yeast/fungal strains were found in Koji, a fermentation starter, and changes in these communities were observed according to fermentation time, and it was reported that the quality of Koji varies depending on the distribution of these strains. In China, the salt-resistant yeasts Z. rouxii and Candida Using versatilis as a spawn of soy sauce, we are conducting basic research to investigate the mechanism of salt resistance as well as applied research to improve taste and flavor.

Various ohmic analysis studies such as genomes, transcripts, proteomics, and metabolites for paste-derived microbial strains have recently begun to attract attention in Korea, but in most cases, some researchers have studied meta-metabolite analysis of intestinal microbial populations. Currently, studies on functional analysis through genome and transcriptome analysis of traditional soybean fermented microorganism strains are currently being conducted on prokaryotic microorganisms. Various yeast and fungi species have been isolated from domestic soybean and soy sauce fermentation, but genome analysis studies, which are the basis for systematic physiological activity and multiple ohmics, have not yet been conducted. Aspergillus , a traditional soybean fermentation fungus in Japan oryzae genome analysis was completed, and Aspergillus recently isolated from Koji Research on fermentation microorganisms for use in the actual industry, such as completing the genome analysis of sojae , is continuing. Since each yeast species produces a different taste and aroma, research on securing flavor-related microorganisms and genetic resources through the discovery of new flavoring yeast strains and genome analysis is being actively carried out worldwide, including Japan's Kikkoman company. Meyerozyma , a flavoring yeast Genomic analysis studies on guilliermondii , Z. rouxii , and C. versatilis were conducted, and based on this, a framework was established in which studies on the function and expression control mechanisms of genes related to flame resistance and flavor can be conducted more systematically.

Meanwhile, the relationship between food safety and eating habits and health is a major concern and issue for consumers in modern society. As a result, while yeast in fermented foods can become a new probiotic microorganism, the safety of food yeast is one of the hot topics in recent years, as it can cause infection or negatively impact consumers in other environments. In particular, the generation of harmful substances such as toxins and biogenic amines is closely related to the metabolism and physiological activity of microorganisms, so a close analysis of individual yeast species is required.

In the case of sauces that are currently manufactured and marketed in large quantities by industry, it is necessary to realize various flavor characteristics of traditional sauces because they have a monotonous flavor. In particular, the importance of the development of new domestic native yeast strains that are expected to play a key role in the taste and aroma of soybeans and the development of seeds with improved flavor and function, immune activity induction, and safety including the same is emerging. Therefore, to discover yeast strains that play a key role in traditional soybean fermentation, to comprehensively understand their functions and physiological activities through various ohmic analysis research, which is a cutting-edge science technology, and develop seeds whose functionality and stability are verified based on them. And fermentation process control technology development is needed.

Korean Patent Registration No. 10-1464546 (registered on November 18, 2014)

An object of the present invention relates to the development of novel traditional soybean fermented yeast strains and probiotic yeast strains using the same, and more particularly to the discovery and identification, growth, and growth of Debaryomyces sp. of Debaryomyces sp. in Korean traditional soybeans . The purpose is to provide a method of utilizing traditional soybean fermentation using the genome, flavor profile characteristics and immune activity modulating ability.

In order to solve the above problems, the present invention is entrusted with KACC 93324P, and has excellent salt resistance and flame resistance, and Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) strains are provided.

In addition, the present invention provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a food composition comprising the strain or a culture thereof as an active ingredient.

In the present invention, new yeasts of the genus Debaryomyces are discovered from fungal/yeast colonies derived from traditional soy sauce and miso samples, and are systematically different from Debaryomyces . Two strains of the genus KD2 and C0-11-Y2 were selected. Both yeast strains showed a basophilic feature that grew better in salty conditions, and KD2 in particular showed higher basophilicity. It was judged that there is no possibility as a human infectious pathogen because it cannot grow even if there is salt at 37℃, which is the temperature inside the human body. The KD2 and C0-11-Y2 strains discovered in the present invention through genomic analysis were previously reported Debaryomyces Hanseni ( Debaryomyces hansenii ) It proved to be a new strain distinct from the strain. Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains produced unique and diverse ingredients such as butter flavor, caramel flavor, and cheese flavor. In addition, as a result of evaluating the induction of immune activity of yeast strains in human dendritic cells, in particular, KD2 and C0-11-Y2 strains induce the inflammation-suppressing cytokine IL-10 to a higher level than previously known probiotic yeasts. Observed. Overall, Debaryomyces Hanseni KD2 ( Debaryomyces) has excellent basophilicity and flavor and excellent immune-suppressing cytokine secretion ability. hansenii KD2) strain has been suggested to be developed as a flavor-enhanced enteric fermentation strain and a new probiotic yeast.

1 is a debaryomyces isolated from soy sauce and miso ( Debaryomyces ) Genus strains and Debaryomyces This is a schematic diagram created using the 5.8S rRNA and ITS2 sequences of the genus test strains.
Figure 2 is the final selected debaryomyces in the present invention ( Debaryomyces ) Genus strain C0-11-Y2 and Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) 5.8S rRNA and ITS2 gene base sequence.
3 shows the results of comparative analysis of salt resistance and growth analysis at 37° C. of Debaryomyces sp. derived from Korean traditional soybeans and yeast-derived and standard strain D. hansenii under various salt stress conditions. (A) 3 days of cultivation in a medium containing various salts, (B) 7 days of cultivation in a medium containing 15% NaCl, (C) results of growth analysis at 37°C.
Figure 4 is Meju / Doenjang-derived Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains are presented with the results of polyploid analysis and genome assembly and annotation. (A) Debaryomyces Hanseni KD2 ( Debaryomyces ) through FACS experiment hansenii KD2), polyploid analysis result of strain C0-11-Y2, (B) genome assembly result of KD2 strain, (C) genome assembly result of C0-11-Y2 strain, (D) KD2 and (E) C0-11 -The result of approximate gene annotation based on transcriptome data of the Y2 strain, (F) the result of whole genome assembly of the KD2 strain at the chromosome level referring to the reference genome D. hansenii CBS767 genome.
5 shows the results of analysis of flavor compounds of Debaryomyces hansenii KD2 strain using SPME-GC/MS. (a) a flavor profile of the KD2 strain, (b) a flavor compound detected from the KD2 strain.
6 is a Debaryomyces sp. using SPME-GC/MS. The analysis results of the flavor compounds of the C0-11-Y2 strain are shown. (A) Flavor profile of C0-11-Y2 strain, (B) Flavor compound detected from C0-11-Y2 strain.
Figure 7 is a paste- derived Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains and the results of measurement of the level of expression of the surface differentiation markers of human DC, hDC, which were reacted with human strains. (A) Gating of hDC during FACS analysis (B), (C), (D) Expression of surface differentiation markers CD86, MHC class II, and MHC class I of human DC, hDC, reacted with enteric yeast Degree.
Figure 8 is a paste- derived Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains and reacted with human-derived dendritic cells.

Accordingly, the present inventors report the discovery and identification of two strains KD2 and C0-11-Y2 of the new yeast Debaryomyces sp. isolated from traditional soybeans , and the growth characteristics, genomic characteristics, and flavor profile characteristics. In addition, the possibility of use as probiotic yeast strains and traditional intestinal strains was suggested by analyzing the ability to modulate immune activity.

The present invention provides a strain of Debaryomyces hansenii KD2 ( Debaryomyces hansenii KD2) that is consigned to KACC 93324P and has excellent basophilic and salt resistance.

Preferably, the strain may be isolated from soybean paste, but is not limited thereto.

Preferably, the strain can produce a flavor component compound that improves the flavor of soybean fermented food, and more preferably, the flavor component compound is fluoroacetylene, isovaleraldehyde, (Z)- 2-Butenal ((Z)-2-Butenal), Isobutyl alcohol, Isoamyl alcohol, Styrene, Acetoin, 2-Nonanone , 2-Hydroperoxypentane, 1-Isopropyl-1-butanol, Isobutanoic acid, butanoic acid, phenylacet Aldehyde (Phenylacetaldehyde), 2-Methylhexanoic acid, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate (3-Hydroxy-2,4,4-trimethylpentyl 2 -methylpropanoate), 1-[2-(isobutyryloxy)-1-methylethyl)-2,2-dimethylpropyl 2-methylpropanoate (1-[2-(Isobutyryloxy)-1-methylethyl)-2 ,2-dimethylpropyl 2-methylpropanoate), 1-(2-hydroxy-1-methylethyl)-2,2-dimethylpropyl 2-methylpropanoate (1-(2-Hydroxy-1-methylethyl)-2, 2-dimethylpropyl 2-methylpropanoate), Phenylethyl alcohol, 2-Ethylbutyl alcohol, Acetic acid, Furfural, Propanoic acid, Perfuryl Furfuryl alcohol, 3-(Methylthio)-1-propanol, Pentyl alcohol, Benzaldehyde And diethyl phthalate (Diethyl phthalate) may be any one or more compounds selected from the group consisting of, but is not limited thereto.

Preferably, the strain may induce the production of IL-10, a cytokine that inhibits inflammation, but is not limited thereto.

In addition, the present invention provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.

In the present invention, "probiotics" or "probiotic strain" refers to a strain that acts beneficial to the intestinal environment when ingested and reaches the intestine. In general, lactic acid bacteria are often used as probiotics, but when yeast is used as probiotics, there are other advantages than lactic acid bacteria. For example, probiotics in general are very sensitive to heat and begin to lose their function even when the temperature exceeds 30℃, whereas yeast is not sensitive to temperature because it is actively active even at 37℃. Also, common probiotics can be neutralized by antibiotics, but yeasts are not affected by antibiotics. Lastly, since general probiotics are weak against gastric acid and bile, various methods must be applied to withstand gastric acid and bile, but yeast has a characteristic that it survives regardless of gastric acid and bile.

The probiotic composition may be used in various applications for improving intestinal function. For example, the probiotic composition of the present invention can be used as a'formal' composition because it has an intestinal action. 'Formal use' refers to the use of treating and/or improving all symptoms caused by abnormal fermentation of the intestinal flora of humans or animals. The symptoms include, but are not limited to, diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic enteritis syndrome, and small intestinal microbial hyperplasia.

The probiotic composition may be prepared and administered in various formulations and methods according to methods known in the art. For example, a composition comprising a strain of Debaryomyces hansenii KD2 of the present invention may be mixed with a carrier commonly used in the food/medicine field.

In addition, the present invention provides a food composition comprising the strain or a culture thereof as an active ingredient.

In the case of the food composition of the present invention, there is no particular limitation on the kind of the food. The food to which the strain or its culture can be added is preferably a fermented food, but, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream It can also be added to dairy products, including various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes.

More preferably, in the embodiment of the present invention, the strain or a culture thereof was added to the production of soy sauce or soybean paste.

In the present invention, "culture" refers to a product obtained by culturing a strain in a known liquid medium or solid medium, and is a concept including a strain.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

< Example 1> Debaryomyces sp . Identification and identification of strains

Soy bean paste, and the sample 1 ml (g) PBS (phosphate buffered saline) solution of 10 ^-1 - 10 ^-3 times diluted and then antibiotics (Tetracycline, 10 mg / L; Chloramphenicol, 50mg / L) of YPD (yeast include the extract peptone dextrose) medium and cultured at 30° C. for 3 days to obtain colonies of mold/yeast. The obtained fungal/yeast colonies were PCR amplified of 5.8S rRNA and ITS2 genes of fungi/yeast using ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') primers, followed by sequencing. The sequencing results were blast searched in NCBI, a genome sequence database, and Debaryomyces 11 strains belonging to the genus were isolated (Table 1). In addition, through the phylogenetic tree comparison, the systematically different Debaryomyces Two strains of the genus KD2 and C0-11-Y2 were finally selected (FIGS. 1 and 2 ).

Separation Strain name Frequency of discovery Soy sauce Aspergillius sp. 8/17 Soy sauce Debaryomyces sp . 6/17 Soy sauce Candida sp . 2/17 Soy sauce Lichthemia sp . 1/17 Miso Debaryomyces sp . 5/16 Miso Candida sp . 7/16 Miso Aspergillus sp . 3/16 Miso Lichtheimia sp . 1/16

< Example 2> Debaryomyces sp . Growth characteristics and flame resistance

Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 yeast strains were subjected to a spotting experiment under a given salt stress condition to analyze the salt resistance. 5%, 10%, 15% NaCl, 10%, 15% KCl, 2 M, 2.5 M sorbitol added YPD medium, each strain was serially diluted from OD=1 to 1/10, spotted, and cultured at 28°C. I did. As a result of cultivation for 3 days, when comparing Debaryomyces hansenii KD2 and C0-11-Y2 strains with the standard strain D. hansenii ATCC 36239 strain and the yeast-derived KCTC 27743 strain, paste-derived KD2 And C0-11-Y2 strains showed halophilic characteristics (FIG. 3A). Debaryomyces derived from yeast and intestine compared to standard strains with poor salt resistance The strains were highly salt-resistant, especially Debaryomyces derived from intestines. sp. The salt resistance of the strains was stronger. As a result of culturing 15% NaCl medium for 7 days, D. hansenii derived from yeast KCTC 27743, Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2), C0-11-Y2 strains, in that order, showed strong salt resistance (Fig. 3B). Intestinal Debaryomyces sp. Prepare each strain with OD 1, 10 ^-1 , 10 ^-2 and 10 ^-3 in YPD normal medium and YPD medium with 5% salt (NaCl) added to check whether the strains can grow at 37°C, which is the human body temperature. Then, spotting was performed (Fig. 3C). Saccharomyces cerevisiae used as a control The strain grows better in normal YPD medium than in salt conditions at 37°C, whereas Debaryomyces Hanseni KD2 ( Debaryomyces) derived from the intestine at 37°C. hansenii KD2), C0-11-Y2, and D. hansenii The strain was not grown in both the normal medium and the salt-added medium.

< Example 3> Debaryomyces sp . Dielectric properties

Paste- derived Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains were analyzed using FACS to obtain prior information required for genome assembly and annotation. Polyploid analysis result, Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) strain was analyzed as haploid (H), and C0-11-Y2 strain as diploid (D) (Fig. 4A).

Genomic information analysis was performed by combining the PacBio Sequel system and Illumina HiSeq error correction. First, to secure a high-quality genome for PacBio SMRT (Single Molecule, Real-Time) sequencing, Debaryomyces Hansen KD2 ( Debaryomyces) hansenii KD2) and C0-11-Y2 strains were treated with lyticase, and the genome was obtained through phenol extraction. As a result of the primary de novo assembly, the genome of the KD2 strain was assembled into 21 contigs, and the size of the genome of the KD2 strain was about 13 Mb (Fig. 4B), and the genome of the C0-11-Y2 strain was assembled into 109 contig, and the size was approximately 26 Mb (Fig. 4C), consistent with the results of the polyploid analysis, the KD2 strain was identified as a haplotype, and the C0-11-Y2 strain was identified as a diploid strain.

Debaryomyces Hansen KD2( Debaryomyces hansenii KD2) and C0-11-Y2 strains were generated and analyzed as a first step for genome annotation, and based on this, the approximate genome shape was found. The KD2 strain has 6,134 protein-encoding genes in a 13 Mb genome, of which 6,063 genes were BLAST-matched with a protein-coding gene known in NCBI (FIG. 4D). The C11 strain has 12,395 protein-encoding genes in the genome of 26 Mb, of which 12,207 genes were BLAST-matched with the protein-coding genes known in NCBI (Fig. 4E). Debaryomyces Hansen KD2( Debaryomyces hansenii KD2) For high-quality genome assembly, whole genome assembly at the chromosome level is performed using various genome combination programs such as Trinity, CANU, Falcon, and Flye, and the genome with reference genome D. hansenii CBS767 using BLAST and genome alignment. Sequence similarity and genome structure were analyzed. The reference strain D. hansenii CBS767 showed 99% identity to the genome sequence, but the genome structure obtained different results, such as the presence of a gap in the translocation and fusion site between chromosomes F and D (Fig. 4F). This suggests that Debaryomyces hansenii KD2 belongs to D. hansenii , but has a strain-specific genomic structure.

< Example 4> Debaryomyces sp . Flavor Profile Characteristics

Debaryomyces isolated from the intestine as yeast strains are known to improve the flavor of fermented soybean food by producing various flavor-related metabolites. sp. The flavor profile of the yeast was analyzed. KD2 and C0-11-Y2 strains were pre-cultured in YPD medium and then inoculated into 25 ml YPD medium with an initial OD of 0.3. After incubation for 0, 12, 24, and 48 hours, 5 ml of each culture solution was transferred to a GC vial, and the flavor component compound was recovered through the SPME headspace, followed by Gas Chromatography-Mass Spectrometry (SPME-GC/MS) analysis. As standard substances, ethyl acetate, isoamyl acetate and three medium chain fatty acids (ethyl butyrate, ethyl hexanoate, ethyl octanoate) were used. SPME-GC/MS conditions are as follows. For Equilibrium, the sample was reacted at 50° C. for 5 minutes. A volatile compound was adsorbed for 30 minutes using an SPME device (a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber), and then eluted at 250° C. for 2 minutes. The eluted compound was injected into a GC-MS instrument (7820A series gas chromatograph-5977E quadrupole mass selective detector, Agilent Technologies, Palo Alto, CA, USA) along a moving line set at 250°C. Separation was performed using an HP-INNOWax GC column (30 m length × 250 μm id × 0.25 μm film thickness) (19091N-133, Agilent Technologies), and the GC conditions are as follows: Oven temperature starts at 40°C, 5 Hold for 10 minutes after increasing the temperature to 150°C at a rate of 5°C/min, and hold for 5 minutes after increasing the temperature to 220°C at a rate of 10°C/min; Carrier gas (He) flow rate 1.0 mL/min; Ionization energy 70 eV; Scan range 33-200 m/z. Each compound was identified using retention indices and mass spectrum data, and mass spectrum data were compared using mass spectral libraries provided by the National Institute of Standards and Technology.

As a result of analysis of the flavor profile of the KD2 strain through SPME-GC/MS (FIG. 5), a large amount of buttery acetoin was detected, and isobutanoic acid and butanoic acid, which have a sour flavor of cheese, also increased with time. Almond and caramel flavored furfural was detected at 24 hours, and almond flavored benzaldehyde was also detected at 48 hours. On the other hand, in the flavor profile of the C0-11-Y2 strain, various peaks did not appear as in the KD2 strain (FIG. 5). Acetoin was detected at 24 hours, but it was detected as a smaller peak than KD2, and isobutanoic acid and butanoic acid were detected similarly to KD2. The styrene giving off unidirectional was detected at slightly higher levels in the C0-11-Y2 strain than in KD2. As a control, a large amount of acetate esters were detected in the flavor profile of the Wickerhamomyces anomalus strain, another intestinal yeast-derived yeast (Figs. 5A and 6A), as a result, Debaryomyces sp. The strains have distinctive flavor profiles, and it was found that in particular the KD2 strain produced a higher level of various important flavor components than the C0-11-Y2 strain.

< Example 5> Debaryomyces sp . Immune activity Controllability

By comparing and evaluating the induction of immune activity of various yeast strains isolated from traditional soybeans, we tried to select yeast seeds with immune enhancing functions that can be developed as probiotics. For the evaluation of probiotics, mononuclear cells adhered to the plate among peripheral blood mononuclear cells (PBMCs) produced from healthy human blood were isolated and cultured in IMDM medium. Count the number of mononuclear cells, 1×10⁶ After adding RPMI medium treated with IL-4 (2 μg/ml) and GM-CSF (5 μg/ml) to the tube to become cells/ml, dispense 2 ml each into a 6-well plate and add 5% CO to 37℃.₂ Incubated in an incubator. The medium was changed every two days and cultured for 6 days to differentiate into immature dendritic cells (iDCs). Immature DCs and yeast strains were reacted to measure the level of expression of surface differentiation markers and cytokine secretion from mature DCs. The yeast strain used was Devariomyces Hanseni KD2 (Debaryomyces hansenii KD2), C0-11-Y2, and is already used as probiotic yeast as a positive control.Saccharomyces boulardii Strain was used. Yeast strains were cultured in 2 ml YPD medium for 16 hours, and then cells were collected and washed with PBS. Measure the OD of yeast cells and use 1×10 RPMI 1640 + 10% FBS medium.⁷It was prepared at a concentration of cells/ml. 1×10 using complete DC medium (RPMI 1640 + 10% FBS) without antibiotics⁶Dispense 100 μl of immature DC prepared at a concentration of cells/ml into a 96-well plate, and add 100 μl of RPMI + 10% FBS as a negative control, and 1×10 as a positive control.⁷cells/ml concentrationS. boulardii Addition of 100 μl of the suspension, Devariomyces Hanseni KD2 (Debaryomyces hansenii KD2) and 100 μl of the C0-11-Y2 strain suspension were added to each of the iDC and yeast strains in an amount of 200 μl at 37℃ 5% CO.₂ It was reacted for 20 hours in an incubator. Each control group and the experimental group were repeatedly tested in 5-well. After the reaction for 20 hours, it was possible to observe that the mononuclear cells were well differentiated into immature dendritic cells, and it was observed that the immature dendritic cells reacted with the yeast strains differentiated into mature dendritic cells.

Cell pellets and supernatant were separated from the yeast-dendritic cell reaction solution by centrifugation in order to observe the expression level of the surface differentiation markers of mature DCs reacted with the yeast strains derived from intestines. The cell pellet was used for differentiation marker analysis, and the supernatant was used for cytokine measurement. For the analysis of differentiation markers, the cell pellet was washed with FACS buffer (2 mM EDTA, 1% BSA, 0.1% sodium azide) and then anti-human HLA-A,B,C (MHC class II) antibodies, anti-human HLA- A solution of DR (MHC class I) antibody and anti-human CD86 antibody was added and reacted at 4° C. for 40 minutes while blocking light. The antibody-stained cells were suspended in 200 μl of FACS buffer and then transferred to a microtube for FACS analysis.

A gating operation was performed to analyze only dendritic cells (FIG. 7A). First, cells were classified by size through FSC-H and SSC-H, and single dendritic cells were classified through FSC-H and FSC-A. Finally, sample analysis was performed by selecting only dendritic cells with CD86 and MHC class II, which are markers of dendritic cells. The measured value was expressed as MFI (mean fluorescence intensity). As a result of CD86 measurement (Fig. 7B), there was no significant difference in CD86 expression from immature dendritic cells in dendritic cells reacted with S. boulardii , which is a positive control. On the other hand, the expression of CD86 was increased in dendritic cells reacted with Debaryomyces hansenii KD2 and C0-11-Y2 strains. This pattern was the same in the MHC class II results (Fig. 7C). On the other hand, the expression of MHC class I did not show a significant difference from immature dendritic cells in all samples (Fig. 7D). Since there was a difference in expression between yeast strains in CD86 and MHC class II, which are known to be specifically highly expressed in dendritic cells, cytokine measurement was performed next.

The Human Cytokines cytometric bead array (CBA) kit was used to measure the amount of cytokines secreted by dendritic cells reacted with yeast. Beads with different sizes and internal fluorescence intensity conjugated with antibodies to IL-1β, IL-6, TNF-α, IL-8, IL12p70, and IL-10 are used as standards for each cytokine or yeast-dendritic phase. It was added to a 96-well V-bottom plate containing the cell culture supernatant and reacted at room temperature for 2 hours. After removal of the supernatant by centrifugation, a biotin-labeled detection antibody was added, reacted at room temperature for 1 hour, and then PE-labeled streptavidin was added and reacted at room temperature for 30 minutes. After removing the supernatant by centrifugation, after adding a wash buffer, the beads were well released by pipetting and transferred to a microtube. After that, FACS analysis was performed. To separate each bead, conditions were set with FSC, SSC, and two lasers (red, blue), and analyzed by setting a flow rate of 25 μl/min and 1800 events (300 events/bead). A standard curve for the amount of each cytokine (0 pg/ml ~ 10,000 pg/ml) was obtained, and a quantitative value of the cytokine secreted by dendritic cells reacted with yeast was obtained using this (Fig. 7). .

Excluding the highest value from each of the five replicates, a graph was drawn against the quantitative value of each cytokine secreted by immature dendritic cells (Fig. 8), and IL-6 and TNF-α showed a meaningful pattern. . S. boulardii compared to immature dendritic cells (iDC) (Sb) induced the inflammation-inducing cytokines IL-6 and TNF-α the highest, and Debaryomyces sp. C0-11-Y2 and KD2 followed. About IL-10, an inflammation-suppressing cytokine that is important in probiotic evaluation because it relieves excessive inflammatory response and is involved in immune tolerance, Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) and C0-11-Y2 strains induced higher levels than S. boulardii . In particular, the KD2 strain induced IL-10 at a level approximately 4 times higher than that of iDC. In previous studies on D. hansenii (Ochangco et al . 2016 , World J Microbiol Biotechnol 32(9):141), strains that induce inflammation-inducing cytokines also induce inflammation-inhibiting cytokines, especially D. hansenii Reported that IL-10 was induced at a higher level than that of S. boulardii , which was consistent with our results. Therefore, Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) was able to confirm the potential as a probiotic yeast.

< Example 6> Devariomyces Hanseni KD2( Debaryomyces hansenii Manufacture of traditional soy sauce and miso using KD2)

The selected soybeans were immersed in water for 14 hours, dehydrated for 2 hours, and cooked at 100°C for 4 hours. Beans (total weight 5.69kg) steamed for 4 hours at 100°C were molded into 6 meju blocks. The molded meju was fermented at 15℃ for 90 days, soaked in 20 ℓ of 11% salt concentration, and then Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) strain was inoculated at a concentration of 1×10 ⁶ /ml and aged at 25°C for 3 months, and then meju (miso) was isolated from salt water (soy sauce). The separated soy sauce was aged for an additional 3 months, and the miso was aged for 9 months to complete.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it will be apparent that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Institute of Agricultural Biotechnology KACC93324P 20190312

Claims (9)

Debaryomyces hansenii KD2 ( Debaryomyces hansenii KD2) strain consigned to KACC 93324P and excellent in basophilic and salt resistance. The method of claim 1, wherein the strain is Debaryomyces hansenii KD2 ( Debaryomyces hansenii KD2) strain, characterized in that isolated from soybean paste. According to claim 1, wherein the strain is Debaryomyces hansenii KD2 ( Debaryomyces hansenii KD2) strain, characterized in that to produce a flavor component compound that improves the flavor of soybean fermented food. The method of claim 3, wherein the flavor component compound is fluoroacetylene, isovaleraldehyde, (Z)-2-butenal ((Z)-2-butenal), and isobutyl alcohol. , Isoamyl alcohol, Styrene, Acetoin, 2-Nonanone, 2-Hydroperoxypentane, 1-isopropyl-1-butanol (1-Isopropyl-1-butanol), Isobutanoic acid, butanoic acid, phenylacetaldehyde, 2-Methylhexanoic acid, 3-hydride Roxy-2,4,4-trimethylpentyl 2-methylpropanoate (3-Hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate), 1-[2-(isobutyryloxy)-1-methylethyl) -2,2-dimethylpropyl 2-methylpropanoate (1-[2-(Isobutyryloxy)-1-methylethyl)-2,2-dimethylpropyl 2-methylpropanoate), 1-(2-hydroxy-1-methylethyl )-2,2-dimethylpropyl 2-methylpropanoate (1-(2-Hydroxy-1-methylethyl)-2,2-dimethylpropyl 2-methylpropanoate), phenylethyl alcohol, 2-ethylbutyl alcohol (2-Ethylbutyl alcohol), acetic acid, furfural, propanoic acid, furfuryl alcohol, 3-(methylthio)-1-propanol (3-( Methylthio)-1-propanol), pentyl alcohol (Pentyl alcohol), benzaldehyde (Benzaldehyde) and any one or more selected from the group consisting of diethyl phthalate (Diethyl phthalate) Debaryomyces hansenii KD2 ( Debaryomyces hansenii KD2) strain, characterized in that the compound. The method of claim 1, wherein the strain induces the production of IL-10, a cytokine that inhibits inflammation, Debaryomyces Hanseni KD2 ( Debaryomyces hansenii KD2) strain. A probiotic composition comprising the strain according to any one of claims 1 to 5 or a culture thereof as an active ingredient. A food composition comprising the strain according to any one of claims 1 to 5 or a culture thereof as an active ingredient. The food composition according to claim 7, wherein the food is a fermented food. The food composition according to claim 8, wherein the fermented food is soy sauce or miso.

Images